1. The Field of the Invention
The present invention relates to systems to separate and collect molecular samples. More specifically, the present invention relates to a system which automatically collects amino acid, protein and nucleic acid samples from gel electrophoresis.
2. Technical Background
Gel electrophoresis is a standard technique used for protein analysis, DNA fragment sizing, and DNA sequencing. Electrophoresis uses an electric field to cause differently charged particles in a sample to migrate at different rates. This difference in rate of migration results in the separation of the particles into bands of identical charge and size. Separation is based on charge and size difference between different molecules. Larger molecules migrate slower through a gel, while smaller molecules migrate more rapidly though a gel. Likewise, molecules which are highly charged migrate a at a faster rate than molecules with a lower charge.
A variety of gel electrophoresis devices are in use. A typical configuration of a device used for gel electrophoresis uses a flat slab of gel between two plates. The gel can be used in either a horizontal or a vertical position. Typically the slab gels have a thickness of about 0.5 mm to about 1.5 mm. The slab gels range from about 8 cm to about 50 cm high and from about 10 cm to about 20 cm wide. Some commercially available slab gel systems use pre-poured gel between disposable plates. Other systems use reusable plates into which a liquid gel is poured and then solidifies through polymer cross-linking or other mechanisms. In addition, pre-formed gels are also available.
Molecules such as proteins, polypeptides, and nucleic acids can be separated to form distinct bands on a slab gel. The particles are separated to a high resolution, meaning that particles with minute differences in charge and/or size are completely separated. A researcher may use gel electrophoresis to determine characteristics of an unknown sample such as size and charge of particles and the number of distinct fragments in the sample. Gel electrophoresis is also used to separate and purify a particular desired protein or nucleic acid from a mixture.
Gel electrophoresis is frequently used to determine the size of a DNA fragment. Nucleic acid molecules such as DNA have a relatively constant charge to size ratio which results in DNA molecules of the same size migrating uniformly in the gel. To determine the size of the DNA molecule of interest, a DNA ladder is mixed with the sample of interest and run on the gel. A DNA ladder consists of DNA fragments of known, but varying lengths. For example a 1,500 base pair (bp) DNA ladder has DNA segments ranging from 100 bp to 1,500 bp in 100 bp increments. The position of the unknown DNA fragment on the gel compared to the DNA ladder can be used to determine the size of the DNA fragment of interest. Prior to running the samples of DNA fragments or proteins on the gel slab, the samples are stained, radioactively labeled, or fluorescently labeled so that the position of the sample bands on the gel can be determined.
The same technique can be used to separate a DNA fragment of interest from a mixture of DNA fragments. Frequently, a researcher is looking for a DNA fragment or other nucleic acid of an approximate length. This desired DNA fragment is obtained in a sample mixed with other DNA fragments. The sample is then mixed with a DNA ladder and separated by gel electrophoresis. The researcher can determine which band on the gel is the desired DNA fragment by the position of the DNA ladder and the bands from the DNA sample. Once the DNA fragment of interest is identified, the band must be removed from the gel for further analysis and use. However, for the separated particle to be used for further analysis and manipulation, the sample bands must be recovered from the gel. Traditionally, removing the bands from the gel has been difficult. The methods used can be labor intensive. Moreover, the sample of interest can be contaminated or otherwise damaged.
Some methods for removing a sample of interest from a gel involve mechanically excising the portion of the gel containing the sample of interest. The sample of interest is then removed from the excised portion of the gel by either chemical or heat extraction methods. The purity of the sample obtained from these methods depend in large part on the skill of the person excising the sample from the gel. The sample of interest may also be denatured by the heat or chemicals used to remove it from the gel. Moreover, the processes take a long time and are not easily automated.
Other methods of manually extracting the sample of interest from the gel have been developed. These methods use electrophoresis to drive the particles into a membrane with a high affinity for DNA placed immediately downstream from the band of interest. The band is then removed from the membrane. Placement of the membrane in the gel can cause a disruption in the electrical field causing the particles to migrate around the membrane which can result in a low collection efficiency or a contaminated sample band.
These and other manual methods of obtaining the sample of interest from a slab gel have many defects. First, most of the methods are highly dependant on the skill of the operator. The methods are also labor intensive which makes the process more time consuming and costly. Moreover, the methods are not easily automated. Also, the methods can result in the contamination of the sample of interest. The heat and chemicals used in some of the methods may also damage the sample of interest.
Accordingly, a need exists for an apparatus that can automatically collect sample bands from a gel electrophoresis system. It would be an additional advancement if the apparatus, could cleanly extract the sample bands. It would be an additional advancement if the accuracy of the device were not dependant on the skill of the operator. It would be a further advancement if the system were capable of simultaneously extracting bands from multiple sample lanes. It would be a further advancement if the system did not use chemicals or temperatures which could damage the sample of interest. It would be an additional advancement if the device could extract a sample band from a slab gel without over diluting the sample. Such an apparatus is described and claimed herein.
The apparatus of the present invention has been developed in response to the present state of the art, and in particular, in response to the problems and needs in the art that have not yet been fully solved by currently available systems and methods for collecting samples from slab gels. Thus, it is an overall objective of the present invention to provide an automatic collection system for collecting samples of interest from a gel electrophoresis slab gel.
To achieve the foregoing objects, and in accordance with the invention as embodied and broadly described herein in the preferred embodiments, an automatic sample collection system for gel electrophoresis is provided. The system is adaptable to be used with a slab gel with one or more lanes and may be retrofitted to existing slab gel systems. A sample of particles such as DNA, RNA, polypeptides, and proteins can be separated into sample bands on the slab gel. The sample collection system can automatically collect one or more of the sample bands. The sample bands can then be further purified and used by an operator of the sample collection system.
As the sample of particles is separated on the slab gel, the sample bands enter a detection zone. A detector is positioned to detect the entry of a particle within the detection zone. The detector can be used to scan all of the lanes of a multiple lane slab gel. Upon detection of the particle, a syringe pump is energized. The syringe pump directs a stream of buffer solution across a gel free zone within a lane of the slab gel. The buffer solution carries the sample band from the gel free zone, through a collection port, and into a collection vial. Generally the buffer solution used is the same buffer solution used in the slab gel. Accordingly, the buffer solution may contain tris-boric acid EDTA hereinafter, TBE, potassium tartrate, tris-acetate EDTA, hereinafter TAE, or other suitable buffers.
The detector of the present system can use a number of methods including UV-Vis absorbance, fluorescence, raman, mass spectrometry; and electrochemical detection. In a presently preferred embodiment, the detector uses fluorescence technology to detect the sample bands. Generally a fluorescent tag is attached to the particles of the sample. A laser can be positioned to excite the fluorescent tags on particles within the detection zone. The laser is selected to have a wavelength that excites the fluorescent tag. Thus when certain fluorescent dyes are used, an argon ion laser with a wavelength of about 488 nm is used.
An optical fiber can be imbedded in the gel to collect fluorescence from the excited particles. The optical fiber is positioned adjacent the sample lanes and within the collection zone. The fluorescence collected by the optical fiber is transmitted through the optical fiber to low-level light detection electronics such as photomultipliers, photodiodes, and CCD cameras. An optical filter may be positioned between the optical fiber and the low-level light detection electronics.
The collection system can be used to simultaneously collect samples from multiple lanes of a slab gel. The laser may be configured to be scanned between multiple lanes of the gel exciting any fluorescently labeled particles within a detection zone of any of the multiple lanes. Any flourescent light from the multiple lanes is transmitted by the optical fibers to low-level light detection electronics. The collection system can distinguish between fluorescence from the multiple lanes based on the position of the laser when the fluorescence is detected.
In another configuration, one or more optical fibers transmit the laser beam from the laser source to one or more detection zones. The one or more optical fibers allow one laser beam to be split and simultaneously be directed on multiple lanes of the slab gel. One or more additional detection optical fibers are positioned to collect any fluorescence from the labeled particles and transmit the light to low-level light detection electronics. An optical fiber switcher can be coupled to the detection optical fibers. The switcher allows the low-level light detection electronics to distinguish between the one or more lanes of the slab gel.
The present invention also relates to a method of collecting a sample band from gel electrophoresis. The method comprises obtaining a sample of interest and fluorescently labeling the sample. The sample is loaded into a gel electrophoresis system with an automatic sample collection system of the present invention. The gel electrophoresis system is activated to separate the particles of the sample of interest into sample bands. A sample band is detected by the sample collection system. A syringe pump is then activated to direct a stream of buffer across a lane of the slab gel. The stream of buffer collects the sample bands and carries the band into a collection vial.
These and other objects, features, and advantages of the present invention will become more fully apparent from the following description and appended claims, or may be learned by the practice of the invention as set forth hereinafter.